Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

Blanco-Romero, Esther; Durán, David; Garrido‐Sanz, Daniel; Rivilla, Rafael; Martín, Marta; Redondo‐Nieto, Miguel

doi:10.1099/mgen.0.000750

Cited by 10 publications

(19 citation statements)

References 94 publications

(204 reference statements)

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“…In F113, AmrZ and FleQ constitute a regulatory hub in which they mutually and transcriptionally repress each other while conversely regulating other functions: AmrZ down-regulates motility and up-regulates putative extracellular matrix (ECM) components production whereas FleQ up-regulates motility and down-regulates putative ECM components production 6 . A similar antagonistic role of both TFs has also been shown by RNA-Seq during rhizosphere colonization 32 .…”

Section: Introductionsupporting

confidence: 68%

“…As previously observed, amrZ mutants are dramatically impaired in the attachment to surfaces and present a different morphology compared to the wild-type strain in the presence of Congo Red (CR) 7 . Since these observations are consistent with a defect in the production of ECM components, we have studied the transcriptomic profile of F113 and its isogenic amrZ mutant using RNA-Seq approach 32 . The RNA-Seq analyses were done under different growth conditions: exponential and stationary phases in liquid cultures and after rhizosphere colonization.…”

Section: Resultsmentioning

confidence: 75%

“…RNA isolation, sequencing, and bioinformatic analysis from F113 and amrZ - cultures grown in SA medium at exponential (OD 600 = 0.6) and stationary phase (OD 600 = 1.2), or from rhizosphere colonization experiments were performed as described in 32 . F113 genes predicted to encode putative extracellular matrix components were selected.…”

Section: Methodsmentioning

confidence: 99%

“…The ECM of F113 is predicted to contain alginate, levan, Pseudomonas acidic polysaccharide (Pap), and poly-β-1, 6-N-acetylglucosamine (PNAG) polysaccharides, functional amyloids in Pseudomonas (Fap), large adhesion protein A (LapA) and medium adhesion protein A (MapA) adhesins, the putative extracellular mannuronan C-5 epimerase PsmE, and the fimbrial low-molecular-weight protein/tight adherence (Flp/Tad) pili 34 . Since the amrZ mutant is defective in biofilm formation 7 and AmrZ has been found as a regulator of some putative ECM components in liquid cultures and rhizosphere 7 , 32 , this work aimed to investigate the role of AmrZ in the regulation of the ECM components in F113. Additionally, we have analyzed the implication of c-di-GMP in this AmrZ-mediated regulation and the impact of this second messenger in the AmrZ-associated phenotypes of biofilm formation, motility, and rhizosphere colonization in F113.…”

Section: Introductionmentioning

confidence: 99%

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Regulation of extracellular matrix components by AmrZ is mediated by c-di-GMP in Pseudomonas ogarae F113

Blanco-Romero

Garrido‐Sanz

Durán

et al. 2022

Sci Rep

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The AmrZ/FleQ hub has been identified as a central node in the regulation of environmental adaption in the plant growth-promoting rhizobacterium and model for rhizosphere colonization Pseudomonas ogarae F113. AmrZ is involved in the regulation of motility, biofilm formation, and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) turnover, among others, in this bacterium. The mutants in amrZ have a pleiotropic phenotype with distinguishable colony morphology, reduced biofilm formation, increased motility, and are severely impaired in competitive rhizosphere colonization. Here, RNA-Seq and qRT-PCR gene expression analyses revealed that AmrZ regulates many genes related to the production of extracellular matrix (ECM) components at the transcriptional level. Furthermore, overproduction of c-di-GMP in an amrZ mutant, by ectopic production of the Caulobacter crescentus constitutive diguanylate cyclase PleD*, resulted in increased expression of many genes implicated in the synthesis of ECM components. The overproduction of c-di-GMP in the amrZ mutant also suppressed the biofilm formation and motility phenotypes, but not the defect in competitive rhizosphere colonization. These results indicate that although biofilm formation and motility are mainly regulated indirectly by AmrZ, through the modulation of c-di-GMP levels, the implication of AmrZ in rhizosphere competitive colonization occurs in a c-di-GMP-independent manner.

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Section: Introductionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regulation of extracellular matrix components by AmrZ is mediated by c-di-GMP in Pseudomonas ogarae F113

Blanco-Romero

Garrido‐Sanz

Durán

et al. 2022

Sci Rep

Self Cite

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“…AmrZ (alginate and motility regulator Z) is a highly conserved transcriptional regulator within the pseudomonads. Initially described as the regulator of the alginate operon ( Baynham et al, 1999 ), it has been characterized as a global regulator essential for environmental sensing and adaptation in P. aeruginosa and P. ogarae F113, where it acts as transcriptional activator and/or repressor of numerous genes ( Jones et al, 2014 ; Martínez-Granero et al, 2014 ; Blanco-Romero et al, 2022 ). In Pto DC3000 AmrZ also acts as a bifunctional regulator, repressing cellulose production and activating alginate production, motility and virulence ( Prada-Ramírez et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

FleQ, FleN and c-di-GMP coordinately regulate cellulose production in Pseudomonas syringae pv. tomato DC3000

Martínez-Rodríguez

López-Sánchez

García-Alcaide

et al. 2023

Front. Mol. Biosci.

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The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels.

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Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly

Roghair Stroud,

Vang,

Halverson

2024

ACS Synth. Biol.

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Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe−microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/P m , LacI/P lac , and AraC/P BAD ) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10−100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe−microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Moreover, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

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Transcriptomic analysis of Pseudomonas ogarae F113 reveals the antagonistic roles of AmrZ and FleQ during rhizosphere adaption

Cited by 10 publications

References 94 publications

Regulation of extracellular matrix components by AmrZ is mediated by c-di-GMP in Pseudomonas ogarae F113

Regulation of extracellular matrix components by AmrZ is mediated by c-di-GMP in Pseudomonas ogarae F113

FleQ, FleN and c-di-GMP coordinately regulate cellulose production in Pseudomonas syringae pv. tomato DC3000

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly

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